Rotaviruses are a major cause of acute, often fatal, gastroenteritis in infants and young children world-wide. Virions contain an 11 segment double-stranded RNA genome. Little is known about the cis-acting sequences and structural elements of the viral RNAs. Using a database of 1621 full-length sequences of mammalian group A rotavirus RNA segments, we evaluated the codon, sequence and RNA structural conservation of the complete genome. Codon conservation regions were found in eight ORFs, suggesting the presence of functional RNA elements. Using ConStruct and RNAz programmes, we identified conserved secondary structures in the positive-sense RNAs including long-range interactions (LRIs) at the 5' and 3' terminal regions of all segments. In RNA9, two mutually exclusive structures were observed suggesting a switch mechanism between a conserved terminal LRI and an independent 3' stem-loop structure. In RNA6, a conserved stem-loop was found in a region previously reported to have translation enhancement activity. Biochemical structural analysis of RNA11 confirmed the presence of terminal LRIs and two internal helices with high codon and sequence conservation. These extensive in silico and in vitro analyses provide evidence of the conservation, complexity, multi-functionality and dynamics of rotavirus RNA structures which likely influence RNA replication, translation and genome packaging.